Clostridia are significant human pathogens and elucidation of the mechanism of ether lipid formation, which differs from that in mammalian tissues may lead to the discovery of new pharmacological agents specific for these bacteria. Work in our laboratory has shown that plasmalogens and their glycerol acetals are characteristic lipids of clostridia and other anaerobic bacteria. In Clostridium butyricum, we have shown that there are three parallel series of phospholipids: the diacyl phosphoglycerides, the alk-1-enyl acyl phosphoglycerides (plasmalogens), and phosphatidylglycerol (PG) acetals of the plasmalogens. The structure of the PG acetal of plasmenylethanolamine, was elucidated by us with the aid of FAB-MS and FAB-CAD-MS/MS. As part of this project, artifacts formed by reaction between the enthanolamine group and the FAB matrix, triethanolamine were characterized. These adducts can interfere with the accurate determination of molecular species composition. Several alternative matrices have been developed which overcome this problem. We have now characterized the second member of this group, the PG acetal of cardiolipin plasmalogen, and have evidence for a third, the PG acetal of plasmenylglycerol. These are minor phospholipids in Clostridia, which may be involved in the biosynthetic pathways to the ether lipids in these bacteria. The ether lipids of clostridia are also involved in the regulation of the bilayer-non-bilayer equilibrium in the cell membrane. Negative FAB-MS and FAB-CAD-MS/MS will be of help in confirming the structures of these lipids and in comparing the molecular species of the various lipid classes.